The dopamine transporter/cocaine receptor (DAT) is the site at which cocaine exerts rewarding/reinforcing effects. Cloning of a rat DAT cDNA by Addiction Research Center scientists during the preceding FY still left open the possibility that multiple dopamine transporter genes or cDNAs could be found. Biphasic Scatchard plots for some cocaine analog binding studies in striatal membrane preparations provided some evidence that such multiple transporters might exist. Most interesting would be mRNAs expressed in midbrain dopamine neurons. Accordingly, cDNAs from a ventral midbrain cDNA library, as well as those from a mouse brain cDNA library that were homologous to the DAT1 cDNA initially cloned in this laboratory were characterized. In a second approach to this problem, binding to COS cells expressing the DAT1 cDNA was characterized to find whether this single expressed cDNA could confer two cocaine analog recognition sites of differing affinities. Ventral midbrain and whole brain cDNA libraries, screened with oligonucleotides complementary to regions most heavily conserved among neurotransmitter transporters, yielded more than 40 transporter-like cDNAs. Several distinct DAT1 dopamine transporter cDNAs, as well as cDNAs encoding GABA, serotonin, choline, and several unidentified members of the neurotransmitter transporter gene family were found. The murine serotonin transporter cDNA localized this gene to the murine homolog of human chromosome 17. Other orphan clones do not transport dopamine or known transporter substrates in Xenopus oocyte assays, while in situ hybridization studies reveal that none is expressed in a dopamine cell-specific fashion. These results support the conclusion that the DAT1 cDNA may represent the single gene encoding the cocaine receptor. DAT1 expression in COS cells can also produce the biphasic Scatchard plots characteristic of the transporter expressed in striatal membranes.